Animal miRNAs play a role in regulating many developmental processes (Kloosterman &Plasterk, 2006) and have been implicated in many human diseases (Garber, 2006). Despite their important roles in development and disease, few targets have been experimentally validated for most microRNAs. This study will focus a single highly conserved microRNA that has been shown to play a role in oncogenesis and tumor suppression in vertebrates, mir-34 (Chang et al., 2007;He et al., 2007), and use several complementary techniques to identify the role of this microRNA in the nematode C. elegans. This study will identify the targets of mir-34 through computational, biochemical, and molecular techniques. In collaboration with the Han and Ding labs, I have developed a computational target prediction method, which scores microRNA binding sites based on features enriched in biochemically purified microRNA targets (Hammell et al., Submitted 2008). This study will build on the results of that computational study by focusing on one microRNA, mir-34, and exploring its functional role in C. elegans through identifying functional targets and the phenotypic consequences of misregulating these target genes. Since mir-34 has no family members in C. elegans yet still has no overt phenotype, I will also computationally identify and genetically validate microRNAs that cooperate to regulate mir-34 target genes. This hypothesis of cooperating microRNAs is supported by preliminary evidence showing that a mir-34 mutant does have overt phenotypes in a sensitized background where one of the C. elegans Argonaute genes has also been deleted. I will test the top 10 predicted targets of this microRNA by constructing translational reporters for the top predicted genes from our computational method. I will biochemically purify targets of mir-34 by immuno- precipitating (IP) proteins in the RNA induced silencing complex (RISC) in a mir-34 mutant background. Zhang et al. (2007) has shown that a RISC-IP method followed by microarray hybridization of co-IP mRNA can successfully recover thousands of microRNA target mRNAs in C. elegans. Repeating the RISC-IP method in worms mutant for mir-34 should result in the loss of mir-34 targets from the pool of RISC- associated mRNA. Finally, I will use genetic epistasis analysis to identify whether mir-34 is sufficient to regulate its identified targets or whether other microRNAs are cooperating to regulate some targets in the absence of mir-34 function. Any information gleaned from this study can also feed back into the target prediction analysis to improve our methods.